Heme binding compounds and use thereof

ABSTRACT

Inhibitors of nitric oxide formation from arginine useful for treating hypotension, inflammation, stroke and to restore vascular contractile sensitivity to the effects of α 1  adrenergic agonists are physiologically active compounds including N.sup.δ -substituted ornithine or N.sup.ε -substituted lysine moieties or monoalkyl carbon-substituted N.sup.δ -substituted ornithine or N.sup.ε -substituted lysine moieties, having the formula ##STR1## wherein R is (CH 2 ) y  CH 3  or H, R&#39; is CH 2  or C(H) (CH 2 ) y  CH 3 , and R&#34; is CH 2  or C(H) (CH 2 ) y  CH 3 , with y ranging from 0 to 5, and x is 0 or 1 and wherein none or only one of R, R&#39; and R&#34; provides an alkyl substituent on ornithine or lysine moiety, and wherein Q is a heme binding moiety and/or a sulfur-containing binding moiety and Q&#39; is --NH 2  when there is a double bond between the omega carbon and Q and Q&#39; is ═NH when there is a single bond between the omega carbon and Q, and physiologically acceptable acid addition salts thereof.

TECHNICAL FIELD

The invention is directed to novel inhibitors of biological nitric oxideformation.

BACKGROUND OF THE INVENTION

For several decades nitroglycerin has been administered to humans as avasodilating agent in the treatment of cardiovascular disease. It hasbeen shown that nitroglycerin so administered is converted in the bodyto nitric oxide which is the pharmacologically active metabolite.Recently, nitric oxide has been shown to be formed enzymatically as anormal metabolite from arginine in vascular endothelium to provide animportant component of endothelium-derived relaxing factors (EDRFs)which are currently being intensively studied as participating inregulation of blood flow and vascular resistance. Macrophages have alsobeen shown to produce nitric oxide in the body as a component of theircell killing and/or cytostatic function.

More recently it has been established that the enzyme forming nitricoxide from arginine, i.e., nitric oxide synthase, occurs in at least twodistinct forms, namely a constitutive form and an inducible form. Theconstitutive form is present in normal endothelial cells, neurons andsome other tissues. Formation of nitric oxide by the constitutive formin endothelial cells is thought to play a role in normal blood pressureregulation. The inducible form of nitric oxide synthase has been foundto be present in activated macrophages and is induced in endothelialcells and vascular smooth muscle cells, for example, by variouscytokines and/or microbial products. It is thought that in sepsis orcytokine-induced shock, overproduction of nitric oxide by the inducibleform of nitric oxide synthase plays an important role in the observedlife-threatening hypotension. Furthermore, it is thought thatoverproduction of nitric oxide by the inducible form of nitric oxidesynthase is a basis for insensitivity to clinically used pressor agentssuch as α₁ adrenergic agonists in the treatment of septic orcytokine-induced shock patients. Moreover, it is thought thatoverproduction of nitric oxide by inducible form of nitric oxidesynthase is involved in inflammation incident to an immune response.

SUMMARY OF THE INVENTION

It is an object of one embodiment of the invention herein to providenovel arginine or citrulline antagonists which inhibit constitutive formof nitric oxide synthase or inducible form of nitric oxide synthase orboth by a binding preferably involving binding to heme cofactor ofnitric oxide synthase (i.e., the catalytically important heme cofactorenfolded in a nitric oxide synthase molecule) as a result of including aheme binding moiety.

The novel compounds herein are physiologically active compoundsincluding N.sup.δ -substituted ornithine or N.sup.ε -substituted lysinemoieties or monoalkyl carbon-substituted N.sup.δ -substituted ornithineor N.sup.ε -substituted lysine moieties, having the formula ##STR2##wherein R is (CH₂)_(y) CH₃ or H, R' is CH₂ or C(H) (CH₂)_(y) CH₃, and R"is CH₂ or C(H) (CH₂)_(y) CH₃, with y ranging from 0 to 5, and x is 0 or1 and wherein none or only one of R, R' and R" provides an alkylsubstituent on ornithine or lysine moiety, and wherein Q is a hemebinding moiety and/or a sulfur-containing binding moiety, and Q' is--NH₂ when there is a double bond between the omega carbon and Q, and Q'is ═NH when there is a single bond between the omega carbon and Q andphysiologically acceptable acid addition salts thereof.

Preferably, the moiety Q contains a thiono, sulfide or sulfhydryl sulfuratom.

Preferred compounds are L-thiocitrulline, L-homothiocitrulline, N.sup.δ-(2-thienyl)methylimino-L-ornithine, and N.sup.ε-(2-thienyl)methyl-L-lysine.

For compounds where Q' is ═NH, physiologically acceptable acid additionsalts are, e.g., acetate, hydrochloride, sulfate, phosphate, succinate,citrate and propionate.

The term "physiologically active" refers to L-enantiomer whether pure orin admixture with D-enantiomer. The D-enantiomers are notphysiologically active. Thus, in the D,L-compounds only the L-enantiomerportion is physiologically active.

Preferably the compounds are pharmaceutically pure, i.e., more than 99%by weight pure (on a water free basis) and contain from 99% to 100% byweight of L-enantiomer (on an L- and D-enantiomer basis).

It is an object of another embodiment herein to provide methods forprophylaxis or treatment of a subject for systemic hypotension orexpected systemic hypotension caused by pathological overproduction ofnitric oxide from arginine by nitric oxide synthase induced in vascularsmooth muscle or endothelial cells in said subject by a cytokine or by abacterial endotoxin. One of these methods comprises administering tosaid subject of a therapeutically effective amount of a compound of theinvention herein. Another of these methods comprises administering tosaid subject a conventional amount of at least one α₁ adrenergic agonistand an amount of compound of the invention herein effective to restorevascular contractile sensitivity to the effects of said α₁ adrenergicagonist.

It is an object of another embodiment herein to suppress an immuneresponse in a subject in need of said suppressing, e.g., where theimmune response is part of an inflammatory response. This methodcomprises administering to a subject in need of said suppressing of animmunosuppressive effective amount, e.g., an inflammation amelioratingamount of compound of the invention herein.

It is an object of still another embodiment herein to provide a methodof prophylaxis or treatment of a subject for a stroke. This methodcomprises administering to said subject of a therapeutically effectiveamount, e.g., a neuronal cell protecting amount, of a compound of theinvention herein.

The term "subject" is used herein to mean any mammal, including humans,where nitric oxide formation from arginine occurs.

The term "prophylaxis" is used herein to mean to prevent or delay theoccurrence of a condition or to ameliorate the symptoms of a conditionshould it occur compared to where prophylaxis is not carried out.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Lineweaver-Burk plot of the reciprocal of the observedreaction velocity (1/v) versus the reciprocal of the concentration ofsubstrate arginine (1/S) for the Ki determination of Example IV.

FIG. 2 is a graph of product formation versus time depicting results ofExample VI.

FIG. 3 is a graph of product formation versus time depicting results ofExample X.

FIG. 4 is a graph of product formulation versus time depicting resultsof Example XI in respect to rat brain nitric oxide synthase.

FIG. 5 is a graph of product formation versus time depicting results ofExample XI in respect to smooth muscle nitric oxide synthase.

DETAILED DESCRIPTION

We turn now in more detail to the novel compounds herein.

As indicated above, one group of compounds herein consists of thosewhere none of R, R' and R" provides an alkyl substituent on ornithine orlysine moiety. Where this is the case, the compounds herein are thoseincluding ornithine or lysine moieties without monoalkylcarbon-substitution thereon. Ornithine moiety is the case where x equalszero. Lysine moiety is the case where x is 1.

As indicated above, another group of compounds herein consists of thosewhere one of R, R' and R" provides an alkyl substituent on ornithine orlysine moiety. Where this is the case, the compounds herein are thoseincluding monoalkyl carbon-substituted ornithine or lysine moieties.Monoalkyl carbon-substituted ornithine moiety is the case where x equalszero. Monoalkyl carbon-substituted lysine moiety is the case where x is1.

We turn now to the moiety Q. A heme molecule is depicted below, whereiniron may be in the ferrous (Fe⁺²) or in the ferric (Fe⁺³) state.##STR3## A heme binding moiety Q binds to the ferrous or ferric iron inthe heme molecule. This binding is supported by spectral data in ExampleV herein. Preferred heme binding moieties Q contain sulfur. Verypreferably, heme binding moieties Q contain sulfide, sulfhydryl, thionoor thienyl sulfur and the sulfur atom of these binds to the iron. Hemebinding moieties Q besides those containing sulfur include imidazole andsubstituted imidazoles, hydrazino, pyrrole, and selenium. Arginineantagonists heretofore have not included a heme binding moiety; e.g.,N^(G) -methyl-L-arginine does not contain a heme binding moiety.

Very preferably Q is selected from the group consisting of ═S, ##STR4##

Most preferably Q is ═S or thienyl.

Examples of Specific compounds having the structural formula set forthabove where none of R, R' and R" provides an alkyl substituent onornithine or lysine moiety are

(1) Thiocitrulline (where x is zero and Q is ═S). This is prepared asset forth in Example I.

(2) N.sup.δ -2-Thioethylimino-L-ornithine (where x is zero and Q is--CH₂ SH). This may be prepared by reacting L-ornithine with ethyl2-S-benzylthioethylimidate (i.e., C₆ H₅ CH₂ SCH₂ CH═NH(OC₂ H₅)),followed by sodium/liquid ammonia reduction to remove the S-benzylgroup. The required imidate is prepared by reacting2-S-benzylthioacetonitrile with ethanol and HCl. The requiredsubstituted acetonitrile may be obtained by reacting commerciallyavailable 2-chloro- or 2-bromoacetonitrile with benzylmercaptan.

(3) N.sup.δ -3-Thiopropylimino-L-ornithine (where x is zero and Q is--CH₂ CH₂ SH). This may be prepared by reacting L-ornithine with ethyl2-S-benzylthiopropylimidate (i.e., C₆ H₅ CH₂ SCH₂ CH₂,CH═NH(OC₂ H₅)),followed by sodium/liquid ammonia reduction to remove the S-benzylgroup. The required imidate is prepared by reacting3-S-benzylthiopropylnitrile with ethanol and HCl. The requiredsubstituted propylnitrile may be obtained by reacting acrylonitrile withbenzylmercaptan.

(4) N^(G) -2-Thioethyl-L-arginine (where x is zero and Q is --NHCH₂ CH₂SH). This may be prepared by reacting N.sup.α-(tert-butyloxycarbonyl)-δ-(S-methylthiopseudoureido)-L-norvalinetert-butyl ester with S-benzylcysteamine. S-Benzylcysteamine is preparedby reacting cysteamine with benzyl chloride at pH 8.5-9.5; under theseconditions the sulfur is far more reactive than the amino group. Thenorvaline derivative is prepared from N.sup.α-(tert-butyloxycarbonyl)-δ-(thioureido)-L-norvaline tert-butyl ester byreaction with methyliodide to form the S-methylthiopseudouroniumcompound.

(5) N^(G) -2-Methylmercaptoethyl-L-arginine (where x is zero and Q is--NHCH₂ CH₂ SCH₃). This may be prepared by the method outlined above in(4) with the modification that methyl iodide is used in place of benzylchloride in the reaction with cysteamine.

(6) N.sup.δ -(2-Thienyl)methylimino-L-ornithine (where x is zero and Qis ##STR5## This is prepared as set forth in Example III.

(7) N.sup.δ -(2-Thienyl)ethylimino-L-ornithine (where x is zero and Q is##STR6## This may be prepared as described in Example III except that2-(2-thiophene)-acetonitrile is substituted for 2-thiophenecarbonitrile.

(8) Homothiocitrulline (where x is 1 and Q is ═S). This is prepared asset forth in Example II.

(9) N.sup.ε -2-Thioethylimino-L-lysine (where x is 1 and Q is --CH₂ SH).This may be prepared as set forth in (2) above except that L-lysine issubstituted for L-ornithine.

(10) N.sup.ε -2-Thiopropylimino-L-lysine (where x is 1 and Q is --CH₂CH₂ SH). This may be prepared as set forth in (3) above except thatL-lysine is substituted for L-ornithine.

(11) N^(G) -2-Thioethyl-L-homoarginine (where x is 1 and Q is --NHCH₂CH₂ SH). This may be prepared as set forth in (4) above except thatL-lysine is substituted for L-ornithine.

(12) N^(G) -2-Methylmercaptoethyl-L-homoarginine (where x is 1 and Q is--NHCH₂ CH₂ SCH₃). This may be prepared as set forth in (5) above exceptthat L-lysine is substituted for L-ornithine.

(13) N.sup.ε -(2-Thienyl)ethylimino-L-lysine (where x is 1 and Q is##STR7## This may be prepared as set forth in (6) above except thatL-lysine is substituted for L-ornithine.

(14) N.sup.ε -(2-Thienyl)propylamino-L-lysine (where x is 1 and Q##STR8## This may be prepared as set forth in (7) above except thatL-lysine is substituted for L-ornithine.

Examples of specific compounds where one of R, R' and R" provides analkyl substituent on ornithine or lysine moiety are:

(15) α-Methyl-DL-thiocitrulline (where R is CH₃, R' is CH₂ and R" isCH₂, and x is zero and Q is ═S). This compound may be prepared by usingα-methyl-DL-ornithine in place of L-ornithine in Example I.α-Methyl-DL-ornithine is commercially available from Sigma Chemicals,St. Louis, Mo.

(16) β-Methyl-DL-thiocitrulline (where R is H, R' is C(H) (CH₃) and R"is CH₂ and x is zero and Q is ═S). This compound may be prepared byusing β-methyl-DL-ornithine in place of L-ornithine in Example I.β-Methyl-DL-ornithine is prepared by reacting diethylacetamidomalonatewith crotononitrile, followed by catalytic hydrogenation of the nitrilegroup to an amino group. Treatment of that intermediate in boiling acidresults in the deesterification and decarboxylation of the originalmalonate moiety yielding in β-methyl-DL-ornithine.

(17) γ-Methyl-DL-thiocitrulline (where R is H, R' is CH₂ and R" is C(H)(CH₃) and x is zero and Q is ═S). This compound may be prepared by usingγ-methyl-DL-ornithine in place of L-ornithine in Example I.γ-Methyl-DL-ornithine is prepared as described above forβ-methyl-DL-ornithine except methacrylonitrile is substituted forcrotononitrile.

(18) α-Methyl-DL-homothiocitrulline (where R is CH₃, R' is CH₂ and R" isCH₂ and x is 1 and Q is ═S). This compound may be prepared by usingα-methyl-DL-lysine in place of L-lysine in Example II. The α-alkyllysines are prepared from the β-ketonitrile having the structureR--CO--CH₂ --CH₂ --CH₂ --CN, where R is the alkyl group (e.g., methyl)by Strecker amino acid synthesis and reduction of the nitrile group bycatalytic hydrogenation followed by hydrolysis.

(19) β-Methyl-DL-homothiocitrulline (where R is H, R' is C(H) (CH₃) andR" is CH₂ and x is 1 and Q is ═S). This compound may be prepared byusing β-methyl-L-lysine in place of L-lysine in Example II. The β-alkyllysines are prepared in the same way as the β-alkyl ornithines exceptthat γ-bromo-γ-alkyl butyronitrile is used in placed of theβ-substituted acrylonitrile and 1M equivalent of base (e.g., sodiummethoxide) is included in the reaction mixture. Alternative syntheses ofβ-alkyl lysines are well known in the literature.

(20) γ-Methyl-DL-homothiocitrulline (where R is H, R' is CH₂ and R" isC(H) (CH₃) and x is 1 and Q is ═S). This compound may be prepared byusing γ-methyl-L-lysine in place of L-lysine in Example II. The γ-alkyllysines are prepared in the same way as the γ-alkyl ornithines exceptthat β-alkyl-γ-bromobutyronitrile is used in place of the α-substitutedacrylonitrile and 1M equivalent of base (e.g., sodium methoxide) isincluded in the reaction mixture. Alternative synthesis of γ-alkyllysines are well known in the literature.

Syntheses of β-methyl-DL-ornithine, γ-methyl-DL-ornithine,α-methyl-DL-lysine, β-methyl-DL-lysine and γ-methyl-DL-lysine aredescribed in Griffith U.S. patent application Ser. No. 07/889,345.L-Enantiomers can be made by using L-enantiomer reactants. D,L-Compoundscan be made by using D,L-reactants. Admixtures of 50 to 100%L-enantiomer with the remainder being D-enantiomer can be prepared byadmixing L-compound with D,L-compound. Admixtures containing less than50% L-enantiomer can be made by synthesizing D-enantiomer (by startingwith D-compound reactant) and admixing this with L-compound orD,L-compound.

The compounds herein are not biologically active as arginine analogswith respect to protein synthesis. Therefore, there is no possibility ofthese interfering with incorporation of arginine into proteins.

We turn now to the methods herein.

As previously indicated, embodiments herein are directed to methods oftreatment of a subject for systemic hypotension caused by pathologicaloverproduction of nitric oxide from arginine by the enzyme nitric oxidesynthase induced in vascular smooth muscle or endothelial cells in saidsubject with a cytokine or by a bacterial endotoxin. The inducement bycytokines, e.g., gamma-interferon, tumor necrosis factor, interleukin-1or interleukin-2 can occur because of therapy with said cytokines, e.g.,chemotherapeutic treatment with tumor necrosis factor or interleukin-2.In such therapy, the cytokine is administered in conventional amountsfor said therapy. However, whereas the period for administration of saidtherapy is normally limited by the eventual occurrence of severehypotension and vascular leak, the method herein allows concomitantadministration of compound of the invention herein (the term prophylaxisincludes said concurrent administration) to delay or eliminate theoccurrence of these symptoms. The inducement by endotoxin from bacterialinfection or other bacterial toxin is known as septic shock and is theleading cause of death in intensive care units, some 250,000 deaths inone year recently in the U.S. This can be an expected condition in caseswhere the immune system is compromised, e.g., because ofimmunosuppression therapy or in AIDS. Septic shock also occurs inimmunocompetent people.

We turn now to the method herein for prophylaxis or treatment of asubject for systemic hypotension caused by biological overproduction ofnitric oxide from arginine by nitric: oxide synthase induced in vascularsmooth muscle or endothelial cells in said subject with a cytokine or bya bacterial endotoxin wherein the method comprises administering to saidsubject a therapeutically effective amount of a compound of theinvention herein. For treatment of systemic hypotension which is alreadyoccurring, the compound is administered in a blood pressure raisingamount, generally 1 mg/kg to 100 mg/kg for L-enantiomer (preferably 2mg/kg to 20 mg/kg for L-thiocitrulline and 2 mg/kg to 20 mg/kg forL-homothiocitrulline) by a route of administration obtaining a fastresponse, normally parenteral, preferably intravenous. For treatment incases where systemic hypotension is expected (i.e., for prophylaxis,i.e., prevention or delay of the condition occurring or to provideameliorated occurrence of the condition), administration is to provide aplasma level of compound of invention herein sufficient to eliminate ordelay the occurring of the hypotension or to reduce the severity of thehypotension which occurs, generally a plasma concentration ranging from1 μM to 100 μM for L-enantiomer (preferably 10 μM to 50 μM forL-thiocitrulline and 10 μM to 50 μM for L-homothiocitrulline) by a routeof administration which can be parenteral (e.g., intravenous) but alsocan be oral (doses to provide this concentration may be determined byconsidering the half-life of the compounds in the body).

We turn now to the method herein for treatment of a subject for systemichypotension caused by pathological overproduction of nitric oxide fromarginine by nitric oxide synthase induced in vascular smooth muscle orendothelial cells in said subject with a cytokine or by a bacterialendotoxin wherein the method comprises administering to a subject inneed of said treatment of a conventional amount of at least one α₁adrenergic agonist and an amount of compound of the invention hereineffective to restore vascular contractile sensitivity to the effects ofsaid α₁ adrenergic agonists. The α₁ adrenergic agonists are used for thesame purpose now (i.e., to increase blood pressure in a hypotensivepatient) but eventually stop working because of loss of vascularcontractile sensitivity. The α₁ adrenergic agonists are used in the samedosages as they are used now for the same purpose, i.e., in conventionaltherapeutically effective amounts. Suitable α₁ adrenergic agonists areepinephrine, norepinephrine, dopamine, phenylephrine, metaraminol,methoxamine, ephedrine, nephentermine and angiotensin II. Doses fordopamine typically range from 2 μg/kg/min to 50 μg/kg/min. Doses forepinephrine typically range from 0.25 mg to 1.0 mg. Doses fornorepinephrine typically range from 2 μg/min to 4 μg/min and aretypically used if dopamine dose exceeds 20 μg/kg/min. Doses forphenylephrine can range from 0.1 to 10 μg/kg. Doses for angiotensin IIcan range from 0.01 to 1 μg/kg. The route of administration of the mostpopular α₁ adrenergic agonists (epinephrine, norepinephrine anddopamine) is intravenous and for the others the route of administrationis intravenous or in some cases subcutaneous. The compound of theinvention herein is administered in an amount effective to restorevascular contractile sensitivity to the effects of the α₁ adrenergicagonist (i.e., to increase and/or prolong the efficacy of the α₁adrenergic agonists), generally 1 mg/kg to 100 mg/kg for L-enantiomer(preferably 2 mg/kg to 20 mg/kg for L-thiocitrulline and 2 mg/kg to 20mg/kg for L-homothiocitrulline) by a route of administration obtaining afast response, normally parenteral, preferably intravenous.

We turn now to the method herein for suppressing an immune response,e.g., where the immune response is part of an inflammatory response, ina subject in need of said suppressing, said method comprisingadministering to a subject in need of said suppressing animmunosuppressive effective amount of compound of the invention herein.This method may be directed to prophylaxis or treatment of a subject forinflammation, e.g., arising from autoimmune conditions includingrheumatoid arthritis and from host-defense immune mechanisms, e.g.,allograft rejection reactions, caused by immunologically induced nitricoxide production in immune cells, said method involving inhibiting saidnitric oxide production in said cells by administering to a subjectpossibly developing or having such inflammation, a nitric oxidesynthesis inhibiting therapeutically effective amount of compound of theinvention herein. The dosages of L-enantiomer compound herein for use inthis method generally range from 1 mg/kg to 1000 mg/kg (preferably 2mg/kg to 200 mg/kg for L-thiocitrulline and 2 mg/kg to 200 mg/kg forL-homothiocitrulline). Methods of administration include oral,intramuscular, subcutaneous and intravenous. The dosages set forth aboveare daily dosages and are administered for a period of time to causesuppression of immune response and attenuation of inflammation, i.e.,two days or more, e.g., for two to three weeks.

We turn now to the method herein for prophylaxis or treatment of asubject for a stroke wherein the method comprises administering to saidsubject of a therapeutically effective amount of compound of theinvention herein. For a stroke in progress, administration is preferablywithin 6 hours of the onset of the stroke, very preferably within 4hours of the onset of the stroke. Since time is of the essence,administration typically is as soon as practical after diagnosis. Thetherapeutically effective amount is a neuronal cell protecting amount,i.e., an amount which causes increase in neuronal cell survival comparedto where the stroke is untreated. Generally, the dose for L-enantiomerranges from 1 mg/kg to 100 mg/kg (preferably 2 mg/kg to 20 mg/kg forL-thiocitrulline and 2 mg/kg to 20 mg/kg for L-homothiocitrulline).Administration is by a route offering a fast response, e.g., parenteral,preferably intravenous or intraarterial. Prophylaxis involves treatmentof those of high risk for a stroke because of medical history andadministration is that amount sufficient to provide an uninterruptedplasma level of compound herein in a neuronal cell protectingconcentration, generally 1 μM to 100 μM for L-enantiomer (preferably 10μM to 50 μM for L-thiocitrulline and 10 μM to 50 μ M forL-homothiocitrulline) and administration is preferably carried outorally on a daily basis.

Dosages are given above for the L-enantiomer. For admixtures ofL-enantiomer and D-enantiomer, dosages are calculated by dividing thosegiven above by the weight percent of L-enantiomer in the admixture.

The invention is illustrated in the following examples.

EXAMPLE I Synthesis of L-Thiocitrulline

N.sup.δ -(Benzyloxycarbonyl)-L-ornithine tert-butyl ester was preparedaccording to a general published procedure (Bodanszky, M.; Bodanszky,A.; The Practice of Peptide Synthesis; Spring Verlag: New York, 1984).Specifically, N.sup.δ -(benzyloxycarbonyl)-L-ornithine (10.0 gm, 37.6mmol, Sigma Chemicals, Inc.) was mixed with tert-butyl acetate (564 ml)and perchloric acid (5.91 ml, 69-72% aqueous solution) at roomtemperature. The reaction mixture became homogenous after 15 minutes andwas stirred at room temperature for 2 days. Water (300 ml) was added tothe reaction mixture, and the two layers obtained were separated. Theorganic layer was extracted twice (2×200 ml) with water, and thecombined aqueous layers were adjusted to pH 9.5-10.0 with 50% NaOH. Theaqueous layer was then filtered to remove a small amount of insolublematerial, and the filtrate was extracted with ethyl acetate; (3×300 ml).The combined ethyl acetate extracts were dried over MgSO₄ and wereconcentrated by rotary evaporation at reduced pressure to yield 7.5 g ofN.sup.δ -(benzyloxycarbonyl)-L-ornithine tert-butyl ester as an oil (65%yield). ¹ H NMR (DCCl₃) δ1.44 (s,9H), 1.5-1.9 (m,4H), 3.23 (t,2H), 3.31(t,1H), 5.09 (s,2H and s,1H broad), 7.35 (s,5H).

N.sup.α -(tert-Butyloxycarbonyl)-N.sup.δ-(benzyloxycarbonyl)-L-ornithine tert-butyl ester: N.sup.δ-(Benzyloxycarbonyl-L-ornithine tert-butyl ester (7.50 g, 23.3 mmol) wasdissolved in 27 ml of methylene chloride, and the solution was cooled to0° C. To that solution was added dropwise tert-butyl pyrocarbonate (5.87g, 26.9 mmol) in 10 ml of methylene chloride. The reaction mixture wasstirred at 0° C. for 1 hour, and stirring was continued for 3 additionalhours at room temperature. The solvent was evaporated at reducedpressure and the oily residue was chromatographed on a column of silicagel (25 cm×30 mm) using hexane:ethyl acetate (3:1) as solvent. Fractionsof approximately 5 ml were collected, and those containing product wereidentified by thin layer chromatography (see below). Chromatographicfractions containing product were pooled and evaporated to a thick oilby rotary evaporation at reduced pressure. The yield was 8.63 gm(93.5%). ¹ H NMR (DCCl₃) δ1.44 (s,9H), 1.46 (s,9H), 1.5-1.9 (m,4H), 3.23(t,2H), 4.17 (t,1H), 4.88 (s,1H broad), 5.09 (s,2H and s,1H broad) 7.35(s,5H).

Thin layer chromatography was carried out on silica gel plates usinghexane:ethyl acetate (3:1) as solvent. N.sup.δ(benzyloxycarbonyl)-N.sup.α-(tert-butyloxycarbonyl)-L-ornithinetert-butyl ester chromatographedwith an R_(f) =0.33 and was detected by fluorescence using a hand-heldUV light.

N.sup.α -(tert-butyloxycarbonyl-L-ornithine tert-butyl ester: The esterfrom the previous step (8.63 gm, 20.45 mmol) was dissolved in 50 ml ofmethanol and 10% Pd/C catalyst (0.90 gm) was added. The mixture washydrogenated on a Parr hydrogenator at 20 psi H₂ for 4 hr. Followingreduction, the reaction mixture was filtered over Celite and evaporatedto dryness by rotary evaporation at low pressure to provide N.sup.α-(tert-butyloxycarbonyl)-L-Ornithine tert-butyl ester as an oil. Theyield was 5.88 gm (100%). ¹ H NMR (DCCl₃) δ1.44 (s,9H), 1.46 (s,9H),1.5-1.9 (m,4H), 2.73 (t,2H), 4.17 (t,1H), 5.20 (s,1H broad).

N.sup.α -(tert-butyloxycarbonyl)-δ-(thioureido)-L-norvaline tert-butylester This product was prepared as described by Feldman (Feldman, P. L.;Tetrahedron Lett. 1991, 32, 875-878). N.sup.α-(tert-butyloxycarbonyl)-L-ornithine tert-butyl ester (5.80 gm) wasdissolved in 100 ml chloroform and added to a solution of 5.70 gm ofcalcium carbonate and 2.2 ml of thiophosgene (28.7 mmol) dissolved in100 ml of water. The mixture was stirred vigorously overnight. The nextday the reaction mixture was filtered and the layers were allowed toseparate. The aqueous layer was extracted with chloroform (2×50 ml) andthe combined organic layers were dried (MgSO₄) and concentrated to anoil by evaporation at reduced pressure. The residue was taken up in drymethanol (200 ml) and cooled to 0° C. Ammonia gas was then passedthrough the solution for 20 minutes, and the solution was stirred for 3hours at 0° C. Following reaction with ammonia, the solvent wasevaporated under reduced pressure and the residue was dissolved in ethylacetate:hexane (4:1 ). That solution was chromatographed on a column ofsilica gel (25 cm×30 mm) using the same ethyl acetate/hexane mixture aseluent. Fractions of approximately 5 ml were collected, and thosecontaining product were identified by thin layer chromatography (seeabove, R_(f) =0.30). Product-containing fractions were pooled andevaporated to dryness under reduced pressure to yield N.sup.α-(tert-butyloxycarbonyl)-δ-(thioureido)-L-norvaline tert-butyl ester in70% yield. ¹³ C NMR (DCCl₃) 183.4-180.4 (one carbon), 17.1-6,155-9,82.2-80.0, 53.6-52.5 (one carbon), 44.6-43.1 (one carbon), 30.4, 28.1,27.8, 24.6.

N.sup.δ -(Thioureido)-L-norvaline (L-Thiocitrulline): N.sup.α-(tert-butyloxycarbonyl)-N-(thioureido)-L-norvaline tert-butyl ester wasmixed with a solution of 4N hydrochloric acid in dioxane and kept atroom temperature for 24 hours. A solid precipitate formed. The mixturewas then diluted with ethyl ether (20 ml) and the entire solution wasevaporated to dryness under reduced pressure. Methanol (10 ml) was addedand evaporated at reduced pressure 2 to 3 times to provide N.sup.δ-thioureido-L-norvaline, called L-thiocitrulline, as a white solid. Theyield was 600 mg, 91%. NMR (CD₃ OD) δ1.40-1.90 (m,4H), 3.16 (t,2H), 3.74(t,1H): ¹³ C NMR (CD₃ OD) δ25.40, 28.69, 44.53, 53.47, 171.48; (IR (KBr)cm⁻¹ : 1710, 1635, 1595, 1470, 1390, 1300; mass spectrum, 192 (MH+).

Reference Example I

D-Thiocitrulline was prepared as described for L-thiocitrulline (ExampleI) except that the starting material was N.sup.δ-(benzyloxycarbonyl)-D-ornithine (Sigma Chemicals, Inc.). Starting with5.0 gm of that material the final yield of D-thiocitrulline was 600 mg(29% overall yield).

Example II Synthesis of N.sup.ε -(Thioureido)-L-norleucine(L-Homothiocitrulline)

N.sup.α -(Benzyloxycarbonyl)-L-lysine tert-butyl ester: N.sup.α-(Benzyloxycarbonyl)-L-lysine (5.0 gm, 17.84 mmol) was dissolved in 268ml of tert-butyl acetate containing 2.80 ml of perchloric acid (69-72%aqueous solution). The solution was stirred for 2 days at roomtemperature and then was extracted with water (3×200 ml). The combinedaqueous layers were adjusted to pH 10 with 50% sodium hydroxide. Theaqueous solution was then extracted with ethyl acetate (3×200 ml) andthe organic layers dried over MgSO₄. After filtration to remove MgSO₄,the solvent was removed by evaporation at reduced pressure to provideN.sup.α -(benzyloxycarbonyl)-L-lysine tert-butyl ester as a clear oil(2.16 gm, 36%). ¹ H NMR (CDCl₃) 1.35 (s,9H), 1.40-2.0 (m,6H), 2.60(t,2H), 4.10 (m, 1H), 4.98 (s,2H), 5.29 (bs,1H), 7.35 (s,5H).

N.sup.ε -(thioureido)-N.sup.α -(Benzyloxycarbonyl)-L-norleucinetert-butyl ester: N.sup.α -(benzyoxycarbonyl)-L-lysine tert-butyl ester(2.16 gm, 6.43 mmol) was dissolved in 30 ml of chloroform and added to aprepared solution of calcium carbonate (1.80 g, 18.0 mmol) in 30 ml ofwater containing 0.70 ml of thiophosgene; the mixture was stirred for 6hours at room temperature. The reaction mixture was then filtered andthe layers separated. The aqueous layer was extracted with chloroform(2×30 ml) and the combined organic layers were dried over MgSO₄. Afterfiltration to remove MgSO₄, the dry solution was evaporated underreduced pressure to yield a clear oil. The residue was dissolved in 55ml of methanol and cooled to 0° C. Ammonia gas was passed into thesolution for 15 minutes and the reaction mixture was stirred for 3 hoursat 0° C. The methanol was then evaporated at reduced pressure and theresidue was chromatographed on a column of silica gel (25 cm× 30 mm)using ethyl acetate:hexane (4:1) (R_(f) =0.33) as the eluent. Fractionscontaining product were identified by thin layer chromatography and werepooled. Evaporation of the product-containing fractions yielded N.sup.α-(benzyloxycarbonyl)-N.sup.ε -thioureido-L-norleucine tert-butyl esteras a foamy solid. ¹³ C NMR (CDCl₃) δ182.70-179.6 (one carbon), 171.35,155.99, 135.77, 129.19, 128,85, 127.08, 126.61, 81.92, 68.5-64.65 (onecarbon) 54.73-52.97 (one carbon), 46.30-41.50 (one carbon), 30.10,28.40, 26.73, 25.0.

N.sup.ε -(thioureido)-L-norleucine (L-Homothiocitrulline): N.sup.α-(carbobenzoxy)-ε-thioureido-L-norleucine (1.31 g, 3.32 mmol) wasdissolved in 4N HCl/dioxane (12 ml) and heated to 75° C. for 1 hour andleft at room temperature for 3 hours. The reaction mixture was dilutedwith ether (20 ml) and the solvents were evaporated under reducedpressure. This was repeated two times. Methanol (10 ml) was then addedand the solvent was evaporated under reduced pressure to provide N.sup.ε-thioureido-L-norleucine, called L-homothiocitrulline, as a white solid(900 mg, 93%).

EXAMPLE III Synthesis of N.sup.δ -(2-thienyl)methylimino-L-ornithine

O-Methyl 2-thienylmethylimidate was prepared by mixing2thiophenecarbonitrile (Aldrich Chemicals, 10.0 g, 0.092 mol) withmethanol (3.73 ml, 0.092 mol) and cooling to 0° C. with stirring. HClgas was passed through the solution for 15 minutes, and the reactionmixture was then left in the refrigerator overnight. During storage, asolid white precipitate formed, and it was triturated with ether (50ml). That suspension was filtered, and the while solid was washed withether (2×50 ml) and dried under vacuum over potassium hydroxide. Theyield-of methyl 2-thienylmethylimidate HCl was 13.1 g (80%).

N.sup.δ -(2-Thienyl)methylimino-L-ornithine was prepared by reaction ofthe previously described imidate with the copper salt of L-ornithine.Specifically, L-ornithine HCl (1.69 g, 10 mmol) and cupric acetate (1.0g, 5 mmol) were dissolved in 13 ml of water, and the solution was cooledto 0° C. The pH of the solution was adjusted to 9.65 with sodiumhydroxide. To that stirred solution was added 2-thienylmethylimidate HCl(2.84 g, 16 mmol) in small portions; the pH was kept between 9.5 and 10by addition of small aliquots of 50% sodium hydroxide. The reactionmixture was stirred at 0° C. for 2 hours and then at room temperaturefor 1 hour. The reaction mixture became turbid and a precipitate formed.The pH was adjusted to 7 and 100 ml of water was added to dissolve thesolid. The resulting solution was applied to a column of Dowex-50 (NH₄⁺, 200-400 mesh, 2.5×30 cm) The resin was washed with 500 ml of waterand then with 0.1M ammonium hydroxide (1 L). The product was eluted with5M ammonium hydroxide solution. Two 500 ml fractions were collected andevaporated to a clear oil under reduced pressure. The residue wasdissolved in a minimum amount of water (50 ml) and the pH was adjustedto 7 with HCl. The resulting solution was again concentrated to drynessby rotary evaporation at reduced pressure, and ethanol (20 ml) wasadded. Repeated evaporation of the solution to dryness at reducedpressure resulted in formation of a white solid which gave an NMRspectra consistent with N.sup.δ -(2-thienyl)methylimino-L-ornithine. Theyield was 1.30 g (54%). ¹ H NMR (D₂ O) δ1.65-1.75 (m,4H), 2.84 (t,2H),3.50 (5,1H), 7.06 (m,1H), 7.60-7.70 (m,2H); mass spectrum, 243 (MH+).

EXAMPLE IV Ki of L-Thiocitrulline Determined with the Rat Brain NitricOxide Synthase

To determine the binding affinity of L-thiocitrulline to rat brainnitric oxide synthase, rate measurements were made at severalconcentrations of L-arginine (1 to 10 μM) in the absence or the presenceof L-thiocitrulline (1, 3, or 5 μM). The reaction mixtures wereidentical to those described in Example X except that the final volumewas 50 μl and only a single time point (5 min) was taken. In FIG. 1, theline denoted by filled in triangles is for measurements for 5 μML-thiocitrulline, the line denoted by open triangles is for measurementsfor 3 μM L-thiocitrulline, the line denoted by filled in circles is for1 μM L-thiocitrulline and the line denoted by open circles is for noL-thiocitrulline and the Y-axis is the vertical line extending upwardlyfrom 0. As shown in FIG. 1, increasing concentrations ofL-thiocitrulline caused increasing degrees of inhibition. The graphshown is a Lineweaver-Burk plot in which the reciprocal of the observedreaction velocity (1/v) is plotted versus the reciprocal of theconcentration of substrate arginine (1/S). Results obtained atconcentrations of 1 μM, 3 μM and 5 μM L-thiocitrulline, and resultsobtained in the absence of L-thiocitrulline, were plotted to giveindividual lines as shown. In such a graph, intersection of all lines onthe Y-axis would indicate purely competitive inhibition, whereas theintersection of the lines on the X-axis would indicate purelynon-competitive inhibition. The actual data falls somewhere betweenthese extremes. From the data, it is concluded that L-thiocitrulline isa competitive inhibitor but with atypical binding as evidenced by thefailure of the plot to intersect exactly on the Y-axis. That means itbinds similarly to substrate arginine, i.e., to the same binding site onthe enzyme surface, but has some additional interaction with the enzyme.From the slopes of the line and the points of intersection, it iscalculated that the Ki of L-thiocitrulline is approximately 2 μM.

EXAMPLE V

Demonstration that the Binding of Thiocitrulline to Nitric OxideSynthase (NOS) Causes Spectral Changes Consistent with a DirectInteraction Between Inhibitor and the Iron of the Heme Cofactor

In common with other heme cofactor containing enzymes, both theconstitutive brain NOS and the inducible smooth muscle NOS displayabsorption spectra that reflect the presence of heme, the state of theiron in the heme (i.e., ferrous or ferric iron), and the interaction ofligands with the iron of heme. As isolated and examined in the absenceof L-arginine, both enzymes display a peak absorbance centered near 397nm, a small shoulder at 420 nm, and broad bands of lower extinctioncentered near 538 and 650 nm. All of these absorbencies indicate thepresence of the heme cofactor. These spectral features are similar tothose of 5-coordinate high spin heme proteins such as horseradishperoxidase and high spin-type cytochrome P-450's. When ligands such assubstrate or inhibitors are bound to NOS, the spectra of the hemecofactor can change in a manner which reflects the presence or absenceof direct interaction between the newly bound ligand and the heme iron.Such interactions are detected on the basis of difference spectra. Thatis, in a dual beam spectrophotometer the reference cuvette contains NOSin buffer whereas the sample cuvette contains NOS plus ligand (substrateor inhibitor) in buffer. The spectrophotometer is then set to scan fromhigh to low wavelengths and will report out any differences in theabsorbance of the reference and sample solutions at each wavelengthexamined.

When such studies are carried out with NOS and the substrate L-arginineis added to the sample cuvette, one observes that the sample cuvetteshows increased absorbance at 392 nm and decreased absorbance atwavelengths greater than approximately 410 nm. Such changes areconsistent with the binding of arginine in a manner in which it does notmake direct interaction with the heme iron. Thus, the heme iron in thearginine-loaded enzyme has only 5 ligands. Four attachments are to thepyrrole nitrogens of the heme itself, and a fifth is a coordinationwhich is thought to be to a sulfur belonging to a cysteine residue inthe protein itself. When a similar study was carried out in which 4.5 μML-thiocitrulline was added to a cuvette containing 3.5 μM rat brain NOS,the sample cuvette showed decreased absorption at 392 nm (.sub.Δ ODabout -0.0035) and increased absorption beginning at approximately 420nm reaching a maximum at about 440 nm. At the latter wavelength,absorption increase was approximately 0.008. These changes, which aresimilar but not identical to type II spectra, are interpreted asindicating that the thiocitrulline is making direct contact with theheme iron. Such spectral changes were not observed in a similar studyusing D-thiocitrulline, indicating that the interaction withL-thiocitrulline is stereospecific and thus attributable to theinhibitor binding to the amino acid substrate binding site. Given thatL-thiocitrulline is bound as a substrate and/or product analog, theportion of the molecule in closest proximity to the heme cofactor mustbe the thioureido side chain. Of the possible liganding atoms involvedin the thioureido side chain, only the sulfur was available to makecontact with the heme iron. That is, L-citrulline, which is identical tothiocitrulline except that the sulfur is replaced by oxygen, does notgive spectral changes of the type described. The side chain ofcitrulline contains all of the possible liganding atoms ofthiocitrulline except for the sulfur.

EXAMPLE VI Inhibition of Smooth Muscle Nitric Oxide Synthase byL-Thiocitrulline and Other Compounds

The activity of L-thiocitrulline and other compounds (namelyL-citrulline, D-thiocitrulline and N^(G) -methyl-L-arginine) asinhibitors of vascular smooth muscle nitric oxide synthase wasdetermined in vitro by monitoring the conversion of [¹⁴ C]arginine to[¹⁴ C]citrulline. Smooth muscle nitric oxide synthase, an example ofinduced nitric oxide synthase (iNOS) was obtained as follows: aorticsmooth muscle cells were cultured by explanting segments of the mediallayer of the aortae of adult male Fischer 344 rats. Aortae were removedaseptically and freed of adventitial and endothelial cells by scrapingboth the luminal and abluminal surfaces. Medial fragments (1-2 mm) wereallowed to attach to dry Primaria 25 cm² tissue culture flasks (Falcon;Oxnard, Calif.) which were kept moist with growth medium until cellsemerged. Cultures were fed twice weekly with medium 199 containing 10%fetal bovine serum, 25 mM HEPES, 2 mM L-glutamine, 40 μg/ml endothelialcell growth supplement (Biomedical Technologies; Stoughton, Mass.) and10 μg/ml gentamyocin (GIBCO; Grand Island, N.Y.). When primary culturesbecame confluent, they were passaged by trypsinization. Cells in passage10-15 were seeded at 20,000/well. When the cells became confluent(density of 60-80×10³ cells in a well), the medium was removed bysuction and fresh medium consisting of 200 μ1 of RPMI 1640 (WhittakerLaboratories) containing 10% bovine calf serum, 25 mM HEPES buffer (pH7.4), 2 mM glutamine, 80 U/ml penicillin, 80 μM/ml streptomycin, 2 μg/mlfungizone, 40 ng/ml interleukin-1 and 50 ng/ml interferon-gamma wasintroduced. Interleukin-1 and interferon-gamma are effective inducers ofiNOS.

In a final volume of 200 μl, the reaction mixtures contained thefollowing: 20 mM sodium HEPES, pH 7.15, 0.1 mM EGTA (ethlylene glycolbis(β-aminoethyl ether)-N,N,N',N"-tetraacetic acid), 0.1 mMdithiothreitol, 100 μM tetrahydrobiopterin, 500 μM NADPH, 25 μM FAD, 25μM FMN and 0.03-0.05 units of nitric oxide synthase (1 unit equals theamount of enzyme necessary to convert 1 nmol of arginine to citrullineand nitric oxide per min). The reaction was begun by adding to thereaction mixture 20 μM L-[¹⁴ C]arginine and, except in the case of thecontrol, 100 μM test compound (L-thiocitrulline, L-citrulline,D-thiocitrulline, and N^(G) -methyl-L-arginine). The reaction mixtureswere maintained at 25° C. and at 3.5, 7.0, and 10.5 minutes 50 μlportions were removed from each reaction mixture and added to 200 μl of100 mM sodium HEPES, pH 5.5 containing 5 mM EGTA. The decrease in pHstops the reaction. The reaction mixtures were then placed in a boilingwater bath for 1 minute which caused the protein to precipitate; theprecipitate was removed by centrifugation. A 225 μl portion of thesupernatant was then removed and applied to a small column of Dowex 50(Na+form, 200-400 mesh, 0.5×3.5 mm). L-[¹⁴ C]citrulline, a product ofthe reaction, was eluted from the columns using 2.0 ml of water. Theeluant was collected directly into a scintillation vial, 10 ml ofscintillation fluid (Econo-Safe, Research Products International Corp.)were added and the contained radioactivity was determined by liquidscintillation counting. Knowing the specific activity of the L-argininein the original reaction mixture, it was possible to convert the cpmcitrulline data to pmol product.

As shown in the figure, product formation was plotted as a function oftime (FIG. 2). In FIG. 2 the line denoted by open circles is thecontrol, the line denoted by filled in circles is for the experimentwith 100 μM L-citrulline, the line denoted by filled in triangles is forthe experiment with 100 μM D-thiocitrulline, the line denoted by opensquares is for the experiment with 100 μM N^(G) -methyl-L-arginine andthe line denoted by open triangles is for the experiment with 100 μML-thiocitrulline.

As shown in FIG. 2, in the absence of test compound (20 μM argininecontrol line) product formation was constant over a 10.5 minute periodobserved. Addition of 100 μM D-thiocitrulline or 100 μM L-citrullinecaused no significant inhibition. Addition of 100 μM L-thiocitrullinecaused virtually complete inhibition at all times observed. Addition of100 μM N^(G) -methyl-L-arginine (L-NMA, the prototypic nitric oxidesynthase inhibitor) also caused substantial inhibition, but NMA wassomewhat less effective than L-thiocitrulline.

EXAMPLE VII The Effect of L-Thiocitrulline on Blood Pressure

To test the ability of L-thiocitrulline to block basal nitric oxideformation in vivo, its effects were tested in anesthetizedSprague-Dawley rats. Rats (250-300 gm) were anesthetized with Inactin(100 mg/kg i.p.) and placed on a heated surgical table to maintain bodytemperature of 36.5° C. Femoral arterial and venous catheters wereimplanted (tips were distal to the renal artery) for measurement ofblood pressure and infusion of compounds, respectively. Blood pressurewas measured using a pressure transducer (Cobe, Inc.) connected to amicrocomputer using data acquisition software (AT-CODAS; Data QInstruments, Akron, Ohio). After waiting several minutes to establish astable baseline blood pressure, L-thiocitrulline (20 mg/kg) was given bybolus injection through the venous catheter. The solution administeredwas 100 mM. Changes in systolic, diastolic, and mean arterial pressurewere monitored for approximately 1 hour. Studies were carried out in 4rats.

Initial (i.e., predrug) systolic, diastolic, and mean arterial pressureswere 126±2, 83±2, and 100±3, respectively. Following administration ofL-thiocitrulline, systolic, diastolic, and mean arterial pressures were142±5, 93±4, and 115±6, respectively. There was no significant effect onheart rate. The studies indicate that L-thiocitrulline is an effectivepressor agent in vivo. Based on earlier studies with N^(G)-methyl-L-arginine, the prototypic nitric oxide synthase inhibitor,L-thiocitrulline is viewed as blocking the basal release of nitric oxidefrom vascular endothelial cells. Decreased release of basal nitric oxideremoves an important vasodilatory mediator involved in normal bloodpressure regulation and results in increased vascular tone. Increasedvascular tone (vasoconstriction) increases systemic vascular resistance;blood pressure consequently increases.

EXAMPLE VIII The Effect of L-Thiocitrulline on Blood Pressure

A human is continuously administered interleukin-2 (1×10⁶ units) for 5days. L-Thiocitrulline (2 to 20 mg/kg/day) is administered by continuousinfusion at a rate sufficient to maintain a systolic blood pressure of80-120 mm Hg. The severe hypotension characteristic of the end ofinterleukin-2 therapy is significantly reduced.

EXAMPLE IX The Effect of L-Thiocitrulline on Increasing Response toPressor Agents

Sprague-Dawley rats are injected intraperitoneally with bacteriallipopolysaccharide (a bacterial endotoxin) (15 mg/kg) alone, togetherwith phenylephrine (6 μg/kg), and together with L-thiocitrulline (20mg/kg) and phenylephrine (6 μg/kg). The treatment with the combinationof L-thiocitrulline and phenylephrine significantly reduces the fall inblood pressure from bacterial lipopolysaccharide administration to agreater degree than phenylephrine alone.

EXAMPLE X Inhibition of Brain Nitric Oxide Synthase by L-Thiocitrullineand Other Compounds

The activity of L-thiocitrulline and other compounds (L-citrulline,D-thiocitrulline and N^(G) -methyl-L-arginine) as inhibitors of ratbrain nitric oxide synthase was determined in vitro by monitoring theconversion of [¹⁴ C]arginine to [¹⁴ C]citrulline. Purified rat brainnitric oxide synthase, isolated as described (McMillan, K., Bredt, D.S., Hirsch, D. J., Synder, S. H., Clark, J. E., and Masters, B. S.,Proc. Natl. Acad. Sci. USA Vol 89, pp., 11141-11145, December 1992) wasobtained from Dr. Bettie Sue Masters, The University of Texas healthScience Center at San Antonio, Department of Biochemistry, 7703 FloydCurl Drive, San Antonio, Tex. 78284.

In a final volume of 200 μl, the reaction mixtures contained thefollowing: 20 mM sodium HEPES, ph 7.5, 0.1 mM EGTA, 0.1 mMdithiothreitol, 10 μg/ml calmodulin, 2 mM calcium chloride, 100 μMtetrahydrobiopterin, 100 μg/ml bovine serum albumin, 500 μM NADPH, 25 μMFAD, 25 μM FMN and 0.03-0.05 units of nitric oxide synthase (1 unitequals the amount of enzyme necessary to convert 1 nmol of arginine tocitrulline and nitric oxide per min). The reaction was begun by addingto the reaction mixture 20 μM L-[¹⁴ C]arginine and, except in the caseof the control, 100 μM test compound (L-thiocitrulline,D-thiocitrulline, L-citrulline and N^(G) -methyl-L-arginine). Thereaction mixtures were maintained at 25° C. and at 3.5, 7.0, and 10.5minutes 50 μl portions were removed and added to 200 μl of 100 mM sodiumHEPES, pH 5.5, containing 5 mM EGTA. The decrease in pH and the bindingof calcium by EGTA stops the reaction. The reaction mixtures were thenplaced in a boiling water bath for 1 minute which caused the protein toprecipitate; the precipitate was removed by centrifugation. A 225 μlportion of the supernatant was then removed and applied to a smallcolumn of Dowex 50 (Na+ form, 200-400 mesh, 0.5×3.5 mm). L-[¹⁴C]Citrulline, a product of the reaction, was eluted from the columnsusing 2.0 ml of water. The eluant was collected directly into ascintillation vial, 10 ml of scintillation fluid (Econo-Safe, RPI Corp,Mt. Prospect, Ill.) were added and the contained radioactivity wasdetermined by liquid scintillation counting. Knowing the specificactivity of the L-[¹⁴ C]arginine in the original reaction mixture, itwas possible to convert the cpm citrulline data to pmol product. Asshown in FIG. 3, product formation was plotted as a function of time. InFIG. 3, the line denoted by open circles is the control, the linedenoted by filled in circles is for the experiment with 100 μML-citrulline, the line denoted by filled in triangles is for theexperiment with 100 μM D-thiocitrulline, the line denoted by opensquares is for the experiment with 100 μM N^(G) -methyl-L-arginine andthe line denoted by open triangles is for the experiment with 100 μML-thiocitrulline.

As shown in FIG. 3, in the absence of inhibitor (20 μM arginine controlline), product formation is constant over the 10.5 minute periodobserved. Addition of 100 μM D-thiocitrulline or 100 μM L-citrullinecaused no significant inhibition. Addition of 100 μM L-thiocitrullinecaused virtually complete inhibition at all times observed. Addition of100 μM N^(G) -methyl-L-arginine (L-NMA, the prototypic nitric oxidesynthase inhibitor) also caused substantial inhibition, but L-NMA wassomewhat less effective than L-thiocitrulline.

EXAMPLE XI Inhibition of Brain and Smooth Muscle Nitric Oxide Synthaseby N.sup.δ -(2-thienyl)methylimino-L-ornithine and Other Compound

The activity of N.sup.δ -(2-thienylmethylimino-L-ornithine as aninhibitor of both the constitutive rat brain nitric oxide synthase andthe inducible smooth muscle nitric oxide synthase was tested using theprotocols previously described in Examples X and VI, respectively. Theresults are shown in FIG. 4 and FIG. 5 respectively. In both thesefigures, the line denoted by open circles is the control (20 μMarginine), the line denoted by filled in circles is for 100 μM N^(G)-methyl-L-arginine (L-NMA) and the line denoted by open triangles is for100 μM N.sup.δ -(2-thienyl)methylimino-L-ornithine. As shown in FIG. 4for the study with brain nitric oxide synthase, citrulline was formed ata constant rate in the presence of 20 μM L-arginine and the absence ofinhibitors (control line, open circles). When the reaction mixtures alsocontain 100 μM N.sup.δ -(2-thienyl)methylimino-L-ornithine (opentriangles), the rate of citrulline formation was decreased verysubstantially. For comparison, the extent of inhibition effected by 100μM L-NMA, the prototypic nitric oxide synthase inhibitor, is also shown(solid circles). Although L-NMA is a somewhat more effective inhibitorthan N.sup.δ -(2-thienyl)methylimino-L-ornithine, both compounds areextremely effective inhibitors and can be expected to be useful drugs.FIG. 5 shows similar studies with the smooth muscle nitric oxidesynthesis (iNOS). As with the rat brain enzyme, N.sup.δ-(2-thienyl)methylimino-L-ornithine is an extremely effective inhibitorcomparable to, but somewhat less effective than, L-NMA.

EXAMPLE XII

Both common carotid arteries are ligated in two groups of femaleSprague-Dawley CFY rats. After 10 minutes, ligations are released andflow is again allowed.

In the case of one group, L-thiocitrulline (20 mg/kg) is administered bybolus injection through a venous catheter 30 minutes after occlusion ofthe arteries. In the case of the other group, no therapeutic agent isadministered.

Histological analysis of stroke volume 24 hours following arteryocclusion shows significant reduction in stroke volume for the groupadministered L-thiocitrulline compared to the group receiving notreatment.

EXAMPLE XIII

Sprague-Dawley rats are injected with 0.25 cc air subdermally in thedorsal area in accordance with an air pouch inflammatory model. The ratsin one group are simultaneously administered intraperitoneallyL-thiocitrulline (20 mg/kg) and this administration is repeated every 12hours for 2 weeks. The rats in another group are not administeredL-thiocitrulline. At the end of the two-week period the group of ratsgiven L-thiocitrulline have significantly less inflammation than therats in the other group.

When the same amount of L-homothiocitrulline is substituted forL-thiocitrulline in Examples IV-X, similar results are obtained.

Many variations of the above will be obvious to those skilled in theart. Thus, the invention is defined by the claims.

What is claimed is:
 1. Physiologically active compounds including N.sup.δ -substituted ornithine or N.sup.ε -substituted lysine moieties or monoalkyl carbon-substituted N.sup.δ -substituted ornithine or N.sup.ε -substituted lysine moieties, having the formula ##STR9## wherein R is (CH₂)_(y) CH₃ or H, R' CH₂ or C(H) (CH₂)_(y) CH₃, and R" is CH₂ or C(H) (CH₂)_(y) CH₃, with y ranging from 0 to 5, and x is 0 or 1 and wherein none or only one of R, R' and R" provides an alkyl substituent on ornithine or lysine moiety, and wherein Q is a sulfur-containing heme binding moiety and Q' is --NH₂ when there is a double bond between the omega carbon and Q and Q' is ═NH when there is a single bond between the omega carbon and Q, and physiologically acceptable acid addition salts thereof.
 2. Physiologically active compounds as recited in claim 1 wherein Q is selected from the group consisting of ═S, --CH₂ SH, --CH₂ CH₂ SH, --NHCH₂ CH₂ SH, --NHCH₂ CH₂ SCH₃, ##STR10##
 3. Physiologically active compounds as recited in claim 1 wherein none of R, R' and R" provides an alkyl substituent on ornithine or lysine moiety.
 4. Physiologically active compound as recited in claim 3 wherein Q is --CH₂ SH.
 5. Physiologically active compound as recited in claim 3 wherein Q is --CH₂ CH₂ SH.
 6. Physiologically active compound as recited in claim 3 wherein Q is --NHCH₂ CH₂ SH.
 7. Physiologically active compound as recited in claim 3 wherein Q is --NHCH₂ CH₂ SCH₃.
 8. Physiologically active compound as recited in claim 3 wherein Q is ##STR11##
 9. Physiologically active compound as recited in claim 3 wherein Q is ##STR12##
 10. Physiologically active compound as recited in claim 1 wherein R is CH₃, R' is CH₂ and R" is CH₂, x is zero and Q is ═S .
 11. Physiologically active compound as recited in claim 1 wherein R is H, R' is C(H) (CH₃) and R" is CH₂, x is zero and Q is ═S.
 12. Physiologically active compound as recited in claim 1 wherein R is H, R' is CH₂ and R" is C(H) (CH₃), x is zero and Q is ═S.
 13. L-Thiocitrulline.
 14. L-Homothiocitrulline.
 15. N.sup.δ -(2-Thienyl)methylimino-L-ornithine.
 16. A method of prophylaxis or treatment of a subject for systemic hypotension or expected systemic hypotension caused by pathological overproduction of nitric oxide from arginine by nitric oxide synthase induced in vascular smooth muscle and endothelial cells in said subject with a cytokine or by a bacterial endotoxin, said method comprising administering to said subject of a therapeutically effective amount of a compound of claim
 1. 17. The method of claim 16 wherein the compound is L-thiocitrulline.
 18. A method for treatment of a subject for systemic hypotension caused by pathological overproduction of nitric oxide from arginine by nitric oxide synthase induced in vascular smooth muscle and endothelial cells in said subject with a cytokine or by a bacterial endotoxin, said method comprising administering to a subject in need of said treatment a conventional therapeutically effective amount of at least one α₁ adrenergic agonist and an amount of a compound of claim 1 effective to restore vascular contractile sensitivity to the effects of said α₁ adrenergic agonist.
 19. The method of claim 18 wherein the compound is L-thiocitrulline.
 20. A method of prophylaxis or treatment of a subject for a stroke, said method comprising administering to said subject of a therapeutically effective amount of a compound of claim
 1. 21. The method of claim 20 wherein the therapeutically effective amount is a neuronal cell protecting amount.
 22. The method of claim 21 wherein the compound is L-thiocitrulline.
 23. Physiologically active compounds as recited in claim 1, wherein Q is selected from the group consisting of ═S, --CH₂ SH, --CH₂ CH₂ SH, --NHCH₂ CH₂ SH and --NHCH₂ CH₂ SCH₃. 